A large body of evidence supports the hypothesis that lens opacity is due to crystallin aggregation. To determine possible causes of this aggregation, our current research program has been directed towards identifying all of the major covalent modifications occurring in vivo to human lens crystallins with emphasis on distinguishing between clear and cataractous lenses. More broadly, the goal of this research has been to elucidate the chemistry that initiates or propagates cataract, which includes the chemistry of aging. Our use of mass spectrometry has led to major advances in identifying these covalent modifications. For example, it has been possible to unequivocally distinguish among a variety of age-related modifications that cause the proteins to be more acidic, such as phosphorylation, deamidation and lysine modification, and to readily identify multiple degradation products. All the major sites of modification in all the major gene products of the water-soluble portion of the lens have been identified. The renewal application includes four Specific Aims. Aim 1 will extend our present studies to include identification of covalent modifications to crystallins present in the water-insoluble fractions of clear and cataractous human lenses. Effective pursuit of this goal includes collaboration with Prof. Larry David (Oregon Health Sciences University). Dr. David requests a subcontract for this collaboration in lieu of direct competition with renewal of his R01 grant. In Aims 2-4, we shall explore linkages between known in vivo modifications of human lens crystallins and their propensity to aggregate. Specific Aim 2 will determine the effect of modifications of beta- and gamma- crystallins on their solubility. In Aim 3, new and highly detailed structural information will be obtained about the mechanisms through which alpha-crystallins solubilize gamma- crystallins. Aim 4 will address the effects of specific modifications to alpha-crystallins that inhibit their ability to perform this chaperone function. New analytical methods based on amide hydrogen exchange and lysine reactivity will be used to accomplish Aims 3 and 4.